Translational activation early in mitogenic activation of quiescent human T cells occurs at the level of translation initiation. Although eIF2 alpha mRNA is very well translated in both quiescent and activated T cells, human Go lymphocytes contain very low levels of eIF2 alpha message that are increased more than 50-fold during activation. Neither increased transcription of the eIF2 alpha gene nor changes in the half- life of the message account for this rapid and large increase. We have therefore hypothesized that stabilization of the nuclear precursor sufficient to allow processing and transport to the cytoplasm might account for the large increase seen with mitogenic stimulation. During the course of mapping the promoter of the eIF2 alpha gene by in vitro transcription, we found a divergent transcript that overlaps the eIF2 alpha primary transcript for approximately 450 nucleotides. We have mapped this in vitro transcript by primer extension analysis and find that it maps to a region of the eIF2 alpha gene that contains a consensus to the Initiator element described by Smale, Baltimore, and Reinberg. This initiator sequence is oriented to generate an antisense transcript and mutation or deletion of the element results in a reproducible 10-fold increase in sense expression of a reporter gene, both enzymatically and by Northern blot hybridization. We have further shown that an adjacent sequence binds a factor (IBP) present in K562 nuclear extract. Mutation of either the Inr element or the adjacent binding site for IBP decreases antisense transcription, and therefore increases eIF-2 alpha gene expression. Experiments are currently underway to map the antisense transcript in vivo and to elucidate its role in the post-transcriptional regulation of eIF2 alpha gene during mitogenic activation of human T cells.